NIR light-activated nanocomposites combat biofilm formation and enhance antibacterial efficacy for improved wound healing

The rise of antibiotic-resistant bacteria necessitates the development of novel antibacterial materials. Photothermal therapy (PTT) and photodynamic therapy (PDT), combined, offer a promising strategy against multi-drug resistant bacteria. Metal-based nanoparticles (NPs), such as copper (Cu), silver (Ag), and gold (Au), are attractive candidates due to their unique optical characteristics and ability to interact with adjacent molecules, enhancing photosensitizers in PDT and PTT. Copper, in particular, shows potent bioactivity, low in vivo toxicity, and can be used in wound dressings and implant coatings. While CuO NPs have demonstrated antimicrobial capability, high concentrations are often required. This study aims to address this limitation by synthesizing and characterizing SeTe-CuO NPs, a novel nanocomposite designed to leverage the synergistic effects of PTT and PDT for enhanced antibacterial activity and improved wound healing.

Existing literature highlights the increasing global threat of antimicrobial resistance driven by the overuse of antibiotics. Researchers have explored various nanomaterial-based strategies to combat this challenge, including the use of metal-based nanoparticles for PTT and PDT. Studies have shown the efficacy of copper-based nanoparticles in eradicating bacteria and inhibiting biofilm formation; however, challenges remain in achieving sufficient antibacterial activity at lower concentrations. The combination of PTT and PDT has been shown to be more effective than either therapy alone in killing bacteria, especially those resistant to multiple drugs. This approach has been investigated with various nanoparticles including copper oxide. The use of selenium and tellurium in combination with copper oxide is a novel approach to enhance the antibacterial and wound healing properties of nanoparticles.

SeTe-CuO NPs were synthesized using a one-pot method involving telluric acid, sodium selenite, CTAB, hydrazine, and ascorbic acid. The synthesis and characterization were performed using TEM, EDS, XRD, XPS, FTIR, and Raman spectroscopy. Photodynamic properties were evaluated using electron paramagnetic resonance (EPR) to detect ROS generation (singlet oxygen and hydroxyl radicals). Photothermal properties were assessed by measuring temperature changes upon NIR laser irradiation (808 nm). Antibacterial activity was evaluated using the agar well diffusion method, assessing the zone of inhibition against *E. coli* and *S. aureus*. Antibiofilm activity was determined by measuring biofilm mass after treatment with varying concentrations of SeTe-CuO NPs. Bacterial growth patterns were monitored by measuring OD600. Membrane disruption was investigated using SEM. A live/dead assay using CLSM with DAPI and PI staining was performed to quantify live and dead bacteria. Intracellular ROS generation was measured using DCFH-DA. Cytotoxicity was evaluated using a CCK-8 assay on L929 cells, and hemolysis was assessed using a hemolysis test on mice blood cells. In vivo studies were conducted using a bacterial infected wound healing model in BALB/c mice. Wound healing was assessed by measuring wound closure percentage, and bacterial clearance was evaluated through Giemsa staining and bacterial culture from wound tissues. Histopathological analysis using H&E staining was performed to examine wound tissue regeneration.

SeTe-CuO NPs exhibited effective photodynamic and photothermal properties under NIR irradiation. The NPs demonstrated potent antibacterial activity against both *E. coli* and *S. aureus*, achieving up to 99% bacterial eradication and significant biofilm inhibition (up to 80% reduction for *E. coli* and 70% for *S. aureus* at 100 µg/mL). SEM images revealed significant membrane damage in bacteria treated with SeTe-CuO NPs. The live/dead assay confirmed a large number of dead bacteria in the SeTe-CuO NPs + NIR laser treated group. Intracellular ROS generation was significantly increased in the presence of SeTe-CuO NPs and NIR laser irradiation. Cytotoxicity studies demonstrated excellent biocompatibility of SeTe-CuO NPs with negligible effect on cell viability (over 95% viability even at 100 µg/mL) and low hemolysis (about 5% at 100 µg/mL). In vivo studies showed that NIR-activated SeTe-CuO NPs significantly accelerated wound closure (93% recovery rate after 12 days) and bacterial clearance compared to control and other treatment groups. Histological analysis revealed intact epidermal layers, reduced inflammation, and enhanced re-epithelialization in the SeTe-CuO NPs + NIR laser group. Giemsa staining confirmed a significant reduction in bacterial load in wounds treated with NIR-activated SeTe-CuO NPs.

The results demonstrate the successful synthesis and characterization of SeTe-CuO NPs with combined photothermal and photodynamic properties. The synergistic effect of NIR light activation further enhanced their antibacterial and antibiofilm activities. The superior in vivo wound healing and bacterial clearance observed in the study highlight the potential of SeTe-CuO NPs as a novel therapeutic agent for treating drug-resistant bacterial infections and promoting wound healing. The excellent biocompatibility of the NPs is a crucial factor contributing to their potential for clinical translation. The mechanism of action involves membrane disruption, ROS generation, and the release of copper ions. The findings suggest that SeTe-CuO NPs could significantly reduce the dosage requirements for conventional antibiotics, representing a valuable addition to the armamentarium against multidrug-resistant bacterial infections.

This study successfully synthesized and characterized SeTe-CuO NPs, demonstrating their significant potential as a novel antibacterial agent for wound healing. The combination of photothermal and photodynamic effects, enhanced by NIR light activation, resulted in superior antibacterial activity, biofilm inhibition, and accelerated wound closure in both in vitro and in vivo studies. Future studies should focus on optimizing the synthesis and evaluating the long-term efficacy and safety of SeTe-CuO NPs in larger animal models before clinical translation.

The in vivo study used a relatively small sample size. While the biocompatibility studies showed promising results, further investigation is needed to fully assess the long-term toxicity profile. The specific mechanisms underlying the synergistic interaction between the photothermal and photodynamic effects require more detailed investigation. The study focused on two specific bacterial strains, and future research should evaluate the efficacy against a broader range of pathogens.